GSM7383613: AME-d21, male, rep3 (RRBS); Mus musculus; Bisulfite-Seq
Ground livers were used for genomic DNA isolation using the DNeasy Blood & Tissue Kit (Qiagen) including RNase treatment. DNA quantity and purity were estimated spectrophotometrically. RRBS (n=5 for each age) was performed at the Genomic Sciences and Precision Medicine Center (GSPMC), Medical College of Wisconsin. RRBS libraries were prepared using 250 ng of genomic DNA, digested with Msp1 (New England Biolabs (NEB), R0106M), and purified using QIAquick Nucleotide Removal Kit (Qiagen, 28004). End-repair A tailing was performed (NEB, M0212L) and TruSeq methylated indexed adaptors (Illumina, 15025064) were ligated with T4 DNA ligase (NEB, M0202L). Size selection was performed with AMPure XP beads (Beckman Coulter, A63882). Bisulfite conversion was performed using EpiTect Plus Bisulfite Kit (Qiagen) as recommended by the manufacturer. Following bisulfite treatment, the DNA was purified as directed and amplified using Pfu Turbo Cx HotStart DNA Polymerase (Agilent Technologies, 600414). Library quantification was performed using Qubit dsDNA HS Assay Kit (Life Technologies, Q32854) and the Bioanalyzer DNA 1000 Kit (Agilent Technologies, 5067-1504). The final libraries from RRBS were prepared for sequencing as per the manufacturer's instructions in the Illumina cBot and HiSeq Paired end cluster kit v3. Samples were sequenced at 51 bp paired end reads using Illumina HiSeq 2000 with TruSeq SBS sequencing kit v3.